Garcia-Souza 2017 MiPschool Obergurgl
Assessment of mitochondrial respiratory function in cryopreserved platelets. |
Link: MITOEAGLE
Garcia-Souza LF, Velika B, Sumbalova Z, Gnaiger E (2017)
Event: MiPschool Obergurgl 2017
Peripheral-blood mononuclear cells (PBMC) and platelets (PLT) are potentially powerful models for the diagnosis of mitochondrial respiratory function and dysfunction, offering a minimally invasive approach in comparison to tissue biopsies. Rapid isolation of platelets and respiratory measurement without delay are required to avoid post-blood harvesting stress and cellular activation followed by metabolic alterations. Although it is well known that PLT are sensitive to temperature fluctuations, tending to activate the cells below 18 ºC [1], we considered cryopreservation as a promising strategy to preserve mitochondrial function in PLT comparable to PBMC [2]. Cryopreservation of PLT is successful for the study of inflammatory properties [3,4]. Therefore, the objective of our study was to optimize this cryopreservation protocol for the measurement of respiratory mitochondrial properties of human PLT.
Human blood cells were isolated in 50 ml Leucosep® tubes with 15 ml of Ficoll-Paque™. 18 ml full blood were diluted 1:1 with sterile DPBS and centrifuged at 1,000 g (first centrifugation: 10 min, room temperature, RT, acceleration 6, no brakes). The PBMC-PLT layer was collected and washed with 25 ml DPBS (120 g; 10 min, RT). The supernatant was combined with 5 ml of diluted plasma obtained from the first centrifugation, and EGTA was added at 10 mM final concentration. After centrifugation at 1,000 g (10 min, RT) the cell pellet was washed with 5 ml DPBS containing 10 mM EGTA (1,000 g; 5 min, RT), resuspended with 0.5 ml DPBS containing 10 mM EGTA and the PLT were counted (SYSMEX XN-350). Cells were resuspended in autologous plasma+5% DMSO for cryopreservation. Cryovials were placed in pre-cooled (4 °C) CoolCell® containers and stored at -80 °C. After one, two and four weeks storage, cells were thawed in a 37 °C water bath, diluted in 10 ml of pre-warmed 37 °C medium (DPBS+10 mM EGTA), centrifuged (1,000 g; 10 min, RT) and resuspended in 0.25 ml DPBS+10 mM EGTA. Stock suspensions of cryopreserved or freshly isolated cells were titrated into the 2-ml chambers of the Oxygraph-2k (OROBOROS INSTRUMENTS, Innsbruck, Austria) containing mitochondrial respiration medium MiR05 (OROBOROS MiR05-Kit) or culture medium M199. An extended substrate-uncoupler-inhibitor titration (SUIT) protocol was developed using MiR05 and adding cells (ce) for measuring endogenous ROUTINE respiration (1ce), with sequential addition of pyruvate (2P; exogenous substrate), uncoupler (3U; ETS capacity), glucose (4Glc; Crabtree effect), malate (5M; no stimulation expected in intact cells), rotenone, (6Rot; residual oxygen consumption, ROX, except for stimulation by PM in permeabilized cells), succinate (7S; stimulation of permeabilized cells), digitonin (8Dig; permeabilization of the plasma membrane in the entire cell population, providing a reference level of 100% nonviable cells in the respirometric cell viability test), cytochrome c (8c; test of outer mt-membrane integrity), antimycin A (9Ama; ROX), ascorbate&TMPD (10AsTm; CIV activity plus autooxidation), and azide (11Azd; chemical background to correct for autooxidation).
There was no significant difference in ROUTINE respiration, R, of freshly isolated PLT in M199 and MiR05. Pyruvate addition resulted in a significant increase in R in MiR05. Cold storage stress was detected as an overall decrease in mitochondrial respiration in both media, without a difference in the 1- to 4-weeks cryopreservation groups (MiR05). Flux control ratios (FCR) of cryopreserved and freshly isolated PLT were preserved, suggesting a global decrease in mitochondrial respiration per cell, without qualitative changes detected in our SUIT protocol. Cell viability of cryopreserved platelets decreased by 20% based on succinate respiration normalized by digitonin titration. This is consistent with an over-all loss of mitochondrial function in the non-viable cells, if permeabilization of these cells occurs in high-Ca2+ medium during rewarming at 37 ºC.
These results illustrate that cryopreservation imposes a substantial damage on PLT. Modifications, such as DMSO concentration and freezing medium cell density, will be investigated in order to improve cell viability and improve cryopreservation of mitochondrial respiratory function.
• Bioblast editor: Garcia e Souza LF, Kandolf G
• O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS
Labels: MiParea: Respiration
Stress:Cryopreservation Organism: Human Tissue;cell: Blood cells, Platelet Preparation: Intact cells, Permeabilized cells
Coupling state: ROUTINE, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.
Pathway: S, CIV, ROX
HRR: Oxygraph-2k
Event: C1, Oral
PBMCs
Affiliations
- Garcia-Souza LF(1,2), Velika B(2,3), Sumbalova Z(2,4), Gnaiger E(2,5)
- Inst Sport Science, Univ Innsbruck, Austria
- Daniel Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria
- Dept Medical Clinical Biochem, Fac Medicine, Pavol Jozef Šafárik University Košice, Slovakia
- Pharmacobiochemical Lab, 3rd Dept Internal Medicine, Fac Medicine, Comenius Univ, Bratislava, Slovakia
- OROBOROS INSTRUMENTS, Innsbruck, Austria. - [email protected]
Figure 1
Figure 1.
References and support
- Straub A, Breuer M, Wendel HP, Peter K, Dietz K, Ziemer G (2007) Critical temperature ranges of hypothermia-induced platelet activation: possible implications for cooling patients in cardiac surgery. Thromb Haemost 97:608-16.
- Karabatsiakis A, Böck C, Salinas-Manrique J, Kolassa S, Calzia E, Dietrich DE, Kolassa IT (2014) Mitochondrial respiration in peripheral blood mononuclear cells correlates with depressive subsymptoms and severity of major depression. Transl Psychiatry 4:e397.
- Johnson L, Tan S, Wood B, Davis A, Marks DC (2016) Refrigeration and cryopreservation of platelets differentially affect platelet metabolism and function: a comparison with conventional platelet storage conditions. Transfusion 56:1807-18.
- Johnson L, Coorey CP, Marks DC (2014) The hemostatic activity of cryopreserved platelets is mediated by phosphatidylserine‐expressing platelets and platelet microparticles. Transfusion 54:1917-26.